A significant proportion of cryb/cryb, Rh71/Rh71 adults or cryb/cry01, Rh71/Rh71 adults previously trained under 12h:12h light:dark cycles fail to maintain rhythmicity under constant darkness conditions, while the rhythmic individuals present a significantly longer period, as compared to controls. The defects of cryb, Rh71 double homozygotes to maintain circadian rhythmicity under constant darkness is partially suppressed by the following conditions: the presence of Rh7+tCH322-180G19; the expression of Rh7Scer\UAS.cNa under the control of either Scer\GAL4P2.4.Pdf or Scer\GAL4tim.PE; the expression of Rh7Scer\UAS.T:Zzzz\FLAG under the control of Scer\GAL4P2.4.Pdf; or the expression of Rh3Scer.UAS.cVa, Rh4Scer.UAS.cVa or Rh5Scer.UAS.cVa under the control of Scer\GAL4P2.4.Pdf.
Following a 6 hour delay in their light-dark cycle, cryb Rh52 flies and cryb Rh61 flies show a similar delay in resynchronizing their evening activity peak as cryb flies. Triple mutant cryb Rh52 Rh61 flies resynchronize over a longer period than either the cryb single or double mutants.
Following a 6 hour delay in their light-dark cycle, cryb norpA39 flies show a longer delay in resynchronizing their evening activity peak (5-7 days) compared to cryb flies. The addition of Rh52 or Rh61 to this background extends the time required for resynchronization to beyond 7 days. The addition of both Rh52 and Rh61 to the cryb norpA39 background results in minimal (if any) tendency to resynchronize circadian behaviour.
Following a 6 hour advancement in their light-dark cycle, cryb norpA39 flies show a longer delay in resynchronizing their circadian behaviour (several days) compared to either single mutant. Introducing either Rh52 or Rh61 to this background interferes more strongly with resynchronization, while the quadruple cryb norpA39 Rh52 Rh61 mutant shows the strongest phenotype.
The retrainment of ninaE17 Rh61 cryb to 6 hour delayed green light (472-603nm) and Y-FL (475-724nm) is significantly slower than that of ninaE17 cryb mutants. Indeed some flies do not adapt to the new photoperiod within 7 days.
In contrast to wild-type animals, per01; ; cryb double mutants expressing Scer\GAL80Pdf.PS and Scer\GAL4Mai179 driven perScer\UAS.cBa display robust locomotor activity rhythms, with a long 25.7h period under constant light conditions.
Under constant darkness, cryb Pdf01 double mutants display locomotor rhythms that are similar to cryb mutants, but with a short 22.8h period. Conversely, cryb Pdf01 double mutants display locomotor rhythms that are similar to Pdf01 single mutants under constant darkness, with a fraction of the flies displaying a weak short-period rhythmicity as reported for Pdf01.
Flies expressing sggEP1576 under the control of Scer\GAL4tim.PE in a cryb background have a locomotor activity period of 22.6 hours under constant darkness conditions, which is longer (and closer to that of wild type) than the locomotor activity period of 20.3 hours seen under these conditions in flies expressing sggEP1576 under the control of Scer\GAL4tim.PE in wild-type background.
Under LD conditions eya1;cryb double mutant flies exhibit the typical bimodal activity pattern consisting of M and E activity bouts, as in wild-type. After transfer into LL conditions, eya1;cryb flies remain rhythmic and free-run with a period close to 24 hours (whereas wild-type flies become immediately arrhythmic).
per01; cryb double mutants exhibit a functional clock with a well-defined evening activity peak anticipating `lights off' that is absent in per01 mutants. The evening peak in activity also arrives earlier during the light phase than the wild-type, possibly suggesting an underlying short-period oscillation. tim01;cryb double mutants and per01;tim01;cryb triple mutants do not show any locomotor anticipation of lights off at 18oC, although some residual behaviour is recovered at 29oC. tim01;cryb double mutants and per01;tim01;cryb triple mutants exhibit no change in the phase relationship of the locomotor peak to the lights off signal, as the peak activity moves progressively later as it tracks increasing length light-dark cycles. Hence cryb suppresses the effect of per01 on circadian activity. This is confirmed by the similar behaviour of per01;tim01;cryb to per01;cryb mutants. Under constant darkness or constant light, the rhythmicity of per01;cryb breaks down during the first day. In per01;cryb mutants, brief light pulses administered at 18oC during the night phase at ZT15 and ZT21 in light-dark 12:12 cycles give advances in the phase of locomotor activity. Thus the `clock' of these flies `remembers' a previously administered light pulse, even in light-dark cycles.
Overexpression of 5-HT1BScer\UAS.cYa under the control of Scer\GAL4tim.PE in a cryb homozygous background does not have a discernible effect on the flies light sensitivity due to the strong phenotype produced by the cryb mutation. However, ~87% of the cryb heterozygotes overexpressing 5-HT1BScer\UAS.cYa under the control of Scer\GAL4tim.PE are rhythmic in constant bright light, while only 13% of the control flies are rhythmic under these conditions. Fast Fourier Transform analysis indicates that cryb heterozygous flies overexpressing 5-HT1BScer\UAS.cYa under the control of Scer\GAL4tim.PE have very strong behavioural rhythms in constant bright light, although the average period is lengthened to about 28 hours.
Reducing 5-HT1B expression, through induction of 5-HT1BIR.Scer\UAS, under the control of Scer\GAL4tim.PE, or increasing cry levels through cryScer\UAS.cDa expression, eliminates the strong behavioural rhythms in constant bright light observed in cryb heterozygous flies overexpressing 5-HT1BScer\UAS.cYa under the control of Scer\GAL4tim.PE.
WGMR.PG; cryb flies show anomalous rhythmicity in when kept in constant light, like cryb flies. The longer period of rhythmicity seen when cryb are kept in constant light of high intensity is suppressed when the flies are also mutant for either WGMR.PG, gl60j or norpA36.
so1; cryb double mutants have the same partially penetrant eye phenotype as so1 single mutants. When these mutants have eye remnants, they can re-entrain to to 6 hour phase advances of the LD cycle in green or red light, but not blue light. When these mutants are completely eyeless, they are unable to re-entrain at any wavelength and their evening activity peak occurs after lights off, while flies with eye remnants show a peak of activity two hours before lights off, as do wild-type flies. Unlike so1 single mutants, so1; cryb double mutants rarely show antidromic phase shifting.
gl60j cryb double mutant flies do not entrain at all to 12 hour light-12 hour dark (LD) cycles. At 100 lux, approximately 70% of the flies show free-running behaviour. The average activity shows a small peak after lights off at 100 lux and at 1000 lux virtually all flies react to lights off with a pronounced activity peak. At 1000 lux, the activity level is generally lower during the light phase than during the dark phase. A free-running circadian activity component is present in 81% of the activity records, and after transfer into constant darkness conditions rhythmic locomotor behaviour usually continues from this free-running component and not from the lights-off peak. Approximately 30% of gl60j cryb double mutant flies show poor rhythmicity, even under LD conditions. At 100 lux, about 20% of the flies are arrhythmic, and about 10% show a complex rhythmicity characterised by several small but significant peaks in the periodograms. gl60j cryb double mutants show neither period changes nor any increase in activity level or intervals between morning and evening locomotor activity peaks after transfer from constant darkness to constant light conditions.
In light-dark (LD) conditions pers,cryb double mutant flies show a temperature dependant defect in locomotor activity cycles. At 18oC all flies show a periodic component of about 24 hours (as seen in wild-type) but about 60% of them also display a minor 19 hour component. At 28oC 79% of the rhythmic flies display the endogenous 19 hour period as the main rhythmic component. When pers,cryb double mutant flies are entrained under an light-dark (LD) 10:10 regime - the entrainment breaks down very rapidly during the first two cycles of the new light/dark regime, with their daytime activity advancing by about 90 minutes in each successive day.
Double mutants of cryb with norpA39, neither of which cause entrainment problems alone, exhibit entrainment problems in the initial LD regime. Only 50% of those that entrain to the initial LD cycle were able to synchronize to a new light regime that applies 16-lux blue light. At lower light intensities nearly all of the double mutants fail to entrain to the new LD cycles. Double mutant pers, cryb flies have complex behavior under LD conditions. The double mutant shows both entrained and free running behavioral components. The double mutants are active at different times essentially every day, so their average activity plot shows a monotonic decrease in behavior throughout the daytime, whereas singly mutant pers flies exhibit a pronounced midday peak of activity.
Expression of Dple\cry2Scer\UAS.T:Hsap\MYC under the control of Scer\GAL4tim.PE successfully restores both naive and trained responses to magnetic field in cryb mutants, and it does so in a wavelength-dependent manner.
cryb mutants expressing cryScer\UAS.T:Hsap\MYC under the control of Scer\GAL4tim.PE show significant naive and trained responses to magnetic fields which are similar in both direction and magnitude to those of wild-type flies.
Similarly to wild-type flies, the majority of cryb mutants expressing Scer\GAL4Mai179 driven cryScer\UAS.cEa do not display locomotor activity rhythms under constant light conditions regardless of the presence or absence of Scer\GAL80Pdf.PS in the genotype.
Similarly to wild-type animals, most of the cryb mutants expressing Scer\GAL4tim.PE driven cryScer\UAS.cEa do not display locomotor activity rhythms under constant light conditions regardless of the presence or absence of Scer\GAL80Pdf.PS in the genotype.
Overexpression of cryScer\UAS.cDa, under the regulation of Scer\GAL4tim.PE rescues the cryb phenotype, with the flies becoming desynchronised and arrhythmic after 1 or 2 days. Overexpression of cryΔ5'.Scer\UAS.cDa, under the regulation of Scer\GAL4tim.PE does not rescue the cryb phenotype, with the flies remaining synchronised for about 6 days, exhibiting a long period phenotype before becoming desynchronised.